Antibodies against a-fetoprotein (Afp) (Santa Cruz). Primary antibody was detected using antiIgG coupled to Alexa 488, Alexa 555 or Alexa 546 (Invitrogen, Jackson). Nuclear labeling was performed with ToPro-3 iodide or DAPI (Molecular Probes). Immunofluorescence staining and GFP CPI-455 chemical information expression were visualized with a Leica TCS-SP2 confocal microscope. For quantification of positive cells, clusters were randomly selected from triplicates of two to three independent experiments and the average value 6 SEM was determined.Results Directed Pancreatic Acinar Differentiation of mESC in a Stepwise Fashion through the Regulation of FGF, Follistatin, and Glucocorticoids Signalling PathwaysUp-regulated expression of genes marking endodermal and pancreatic cell populations during stages 1?. Our aimAmylase Secretion AssayCells were washed with PBS and incubated with fresh cell culture medium without FBS and supplemented or non-supplemented (controls) with 1 pM cholecystokinin octapeptide (CCK) (Sigma) or with 5 mM carbachol for 30 min at 37uC. Culture supernatants were then collected and cells lysed in Krebs-Ringer buffer containing 0.2 BSA. Amylase activity was determined using the InfinityTM Amylase Liquid Stable Reagent (Termo Electron). To normalize the amount of amylase secretion, the total protein content was measured by the CY5-SE site Bradford method. Amylase released into the supernatant and amylase content of the cell pellets were determined in triplicates.StatisticsStatistical differences were analyzed by the Student’s t test. p values as *p,0.1; **p,0.05 and ***p,0.005 were considered statistically significant.was to analyze the ability of endoderm enriched ESC populations to respond to specific signals involved in pancreatic development in vivo, using culture conditions previously established to drive mESC into definitive endoderm with minor modifications [33,34]. ESC were aggregated in suspension for one day in low SR concentration (3 ) as EB, to mimic cell interactions occurring in vivo. On the next day, EB were treated with 100 ng/ml activin A for up to 4 days to potentiate endoderm specification (stage 1, Fig. 1). We assessed the expression of early germ-layer specific markers by qRT-PCR. This treatment enhanced the expression of Gsc and T/Bra after 3 days of culture (Fig. 2A ). At day 5, T/Bra was down-regulated while Gsc was further enhanced, suggesting that the cell cultures progress through a transient mesendoderm step. Indeed, from day 1 to day 5, the extra-embryonic endoderm marker Sox7 and the mesoderm marker Myf5 did not show a marked increase, whereas the neuroectoderm markers Sox1 and Zic1 were drastically down-regulated in comparison with nontreated cultures (Fig. 2B ). By contrast, the definitive endodermal markers Foxa2, Cxcr4, Gata4 and Sox17 were significantly upregulated in the treated cultures at day 5 (Fig. 2A), indicating an activation of the definitive endoderm differentiation program. In agreement with endoderm and primitive gut formation, evidenced by the increase in HNF1b and HNF4a (Fig. 2D), the levels of mRNAs encoding for pancreatic Pdx1 were already strikingly enhanced (Fig. 2D), suggesting the activation of a posterior foregut differentiation program. To further promote pancreatic specification, activin A-treated EB were next incubated in suspension during 2 days with FGF10, RA, the hedgehog-signalling inhibitor cyclopamine as previously described [5] and DM, a small-molecule inhibitor of BMP signalling (stage 2, Fig. 1).Antibodies against a-fetoprotein (Afp) (Santa Cruz). Primary antibody was detected using antiIgG coupled to Alexa 488, Alexa 555 or Alexa 546 (Invitrogen, Jackson). Nuclear labeling was performed with ToPro-3 iodide or DAPI (Molecular Probes). Immunofluorescence staining and GFP expression were visualized with a Leica TCS-SP2 confocal microscope. For quantification of positive cells, clusters were randomly selected from triplicates of two to three independent experiments and the average value 6 SEM was determined.Results Directed Pancreatic Acinar Differentiation of mESC in a Stepwise Fashion through the Regulation of FGF, Follistatin, and Glucocorticoids Signalling PathwaysUp-regulated expression of genes marking endodermal and pancreatic cell populations during stages 1?. Our aimAmylase Secretion AssayCells were washed with PBS and incubated with fresh cell culture medium without FBS and supplemented or non-supplemented (controls) with 1 pM cholecystokinin octapeptide (CCK) (Sigma) or with 5 mM carbachol for 30 min at 37uC. Culture supernatants were then collected and cells lysed in Krebs-Ringer buffer containing 0.2 BSA. Amylase activity was determined using the InfinityTM Amylase Liquid Stable Reagent (Termo Electron). To normalize the amount of amylase secretion, the total protein content was measured by the Bradford method. Amylase released into the supernatant and amylase content of the cell pellets were determined in triplicates.StatisticsStatistical differences were analyzed by the Student’s t test. p values as *p,0.1; **p,0.05 and ***p,0.005 were considered statistically significant.was to analyze the ability of endoderm enriched ESC populations to respond to specific signals involved in pancreatic development in vivo, using culture conditions previously established to drive mESC into definitive endoderm with minor modifications [33,34]. ESC were aggregated in suspension for one day in low SR concentration (3 ) as EB, to mimic cell interactions occurring in vivo. On the next day, EB were treated with 100 ng/ml activin A for up to 4 days to potentiate endoderm specification (stage 1, Fig. 1). We assessed the expression of early germ-layer specific markers by qRT-PCR. This treatment enhanced the expression of Gsc and T/Bra after 3 days of culture (Fig. 2A ). At day 5, T/Bra was down-regulated while Gsc was further enhanced, suggesting that the cell cultures progress through a transient mesendoderm step. Indeed, from day 1 to day 5, the extra-embryonic endoderm marker Sox7 and the mesoderm marker Myf5 did not show a marked increase, whereas the neuroectoderm markers Sox1 and Zic1 were drastically down-regulated in comparison with nontreated cultures (Fig. 2B ). By contrast, the definitive endodermal markers Foxa2, Cxcr4, Gata4 and Sox17 were significantly upregulated in the treated cultures at day 5 (Fig. 2A), indicating an activation of the definitive endoderm differentiation program. In agreement with endoderm and primitive gut formation, evidenced by the increase in HNF1b and HNF4a (Fig. 2D), the levels of mRNAs encoding for pancreatic Pdx1 were already strikingly enhanced (Fig. 2D), suggesting the activation of a posterior foregut differentiation program. To further promote pancreatic specification, activin A-treated EB were next incubated in suspension during 2 days with FGF10, RA, the hedgehog-signalling inhibitor cyclopamine as previously described [5] and DM, a small-molecule inhibitor of BMP signalling (stage 2, Fig. 1).